1. Field
The present invention is in the field of microfluidic devices, and relates to a microfluidic device and apparatus for performance of clinical laboratory assays.
2. Description of the Related Art
Clinicians routinely rely on laboratory testing to better assess the condition of patients under their care. Large clinical analyzers such as the “robot chemist” (U.S. Pat. No. 3,193,358), Technicon AutoAnalyzer, and Automatic Clinical Analyzer (Dupont, Wilmington Del.) are well known. A step toward miniaturization of clinical assays is taken in U.S. Pat. No. 3,799,742 to Coleman.
But microfluidic chips have yet greater advantages of reduced scale and are adapted for receiving microliter or nanoliter-sized samples. However, as noted by Lilja in U.S. Pat. No. 4,088,448, mixing of reagents and analytes to homogeneity is more difficult and time consuming in smaller reaction vessels, necessitating the need for some mechanical vibratory means, for example. Lilja reports that the optimal vibrational frequency and amplitude is best determined experimentally. Ultrasonic mixing has also been put forward by Liston (in U.S. Pat. No. 4,528,159), by Yang (Yang Z et al, 2001, Ultrasonic micromixer for microfluidic systems. Sensors Actuators A: Physical 3:266-272), and by Yaralioglu (Yaralioglu GG et al, 2004, Ultrasonic mixing in microfluidic channels using integrated transducers, Anal Chem 76:3694-98). Centrifugation has used for mechanical mixing and dissolution of reagents from reagent discs, as in the Piccolo Chemistry Analyzer (CardinalHealth, Dublin Ohio), which delivers results in about 12 minutes and is described by Schembri (Schembri CT et al, 1992, Portable simultaneous multiple analyte whole-blood analyzer for point-of-care testing, Clin Chem 38:1665-1670). Other mixing means are proposed in U.S. Pat. Nos. 6,382,827 and 6,808,304, and recently by Qian (Qian M et al, 2008, Fabrication of microfluidic reactions and mixing studies for luciferase detection. Anal Chem 80:6045-50), pointing to a difficult and unsolved problem.
Hammond, in U.S. Pat. No. 4,965,047, describes an analytical test strip with frangible blister to hold a diluent or reagent solution. A sample reacts with the liquid reagent in an absorbent layer and endpoint color is read by reflectance in a viewing window. Kitaguchi in U.S. Pat. No. 7,625,760 observes that uTAS methods which utilize reagents supplied from outside the chip where the analysis is performed are well known, such as early proposed by Wilding (U.S. Pat. No. 5,304,487). But noting that this level of complexity is not well suited for point of care analytical applications, Kitaguchi proposes methods for on-cartridge liquid reagent storage and release suitable for quantitative analysis. As observed for example in FIGS. 8 and 13 of the Kitaguchi patent, this method nonetheless adds substantial complexity to the device compositions and is apparently limited to endpoint assays because of the difficulty in instantaneously mixing reagents and analytes.
Subraminian in U.S. Pat. No. 5,223,219 relies on capillary flow and a porous reflective matrix to perform assays for a variety of clinical analytes in blood applied directly to the device. A monitor records the reflectance of the device reaction matrix during the assay reaction, typically at three wavelengths. By comparing the change in reflectance at an endpoint with those for known calibration materials, the monitor can compute the analyte concentration. However, capillary flow has proven difficult to standardize and the irregularities may increase overall assay reproducibility and accuracy.
Oosta in U.S. Pat. No. 5,478,751 describes devices made of self-venting materials in which a reaction such as the glucose oxidase/peroxidase assay for blood glucose is allowed to react to completion in a sample and then the intensity of an indicator dye is read spectrophotometrically. A device body made of microporous polypropylene films, monofilament woven screens, or other hydrophobic, air transmitting materials, is used to eliminate air from the internal channel.
Naka, in U.S. Pat. No. 6,001,307, claims a device for small scale analytical measurements in liquid samples. After the analytical reaction has proceeded for a selected period of time, any resulting pigment is entrapped in a filter paper or sponge in an optical viewing area and measured by densitometry, using reflected light for measuring the production of a pigment Immobilized solid state immunochemistries may also be deployed in the optical viewing area, where a sandwich of analyte and binding reagent is assayed after a defined reaction time. In other words, the assay methods are again limited to endpoint reactions. The inventors also stress the importance of a bypass channel (6) in the claims, which is used so as to take up excess sample or entrained air and neutralize any excess suction pressure (Col 14, lines 25-53, Col 4, lines 40-55), implementing by design diminishing flow resistances greatest in an upstream drawing channel segment, less in a bypass channel, and least in a downstream analytical channel, although this would seem unnecessary in practice.
Naka, in U.S. Pat. No. 6,325,975, further describes an elongate sampling channel affixed to a small thumb-sized suction chamber, the suction chamber having elasticity for generating suction when deformed and released, where the elongate channel contains a second chamber having a “sample analysis device” which contains immobilized reagents for reacting with an analyte. The device has the disadvantage that the sample must be admitted to the device after the suction chamber is depressed, there being no check valve or vent to prevent expulsion of the sample upon application of positive pressure. Also, the incorporation of reagents in a laminated membrane effectively limits reaction rates to diffusional kinetics which are independent of analyte concentration, thus limiting the device to endpoint reactions.
It can be desirable to have results from multiple assays in order to better characterize the clinical status of a patient. Test panels are most helpful if available in real time at the point of care. Challenging are multiple analyses to be performed on sample sizes of 10 or 20 microliters of whole blood, such as obtained with capillary sample tubes. Of particular difficulty are pediatric blood samples, where a high hematocrit is associated with reduced plasma volume of the sample.
Where multiple assays are desired from a minimum sample volume, it may be desirable to perform the assays in series rather than in parallel, metering out the sample only as needed, and in order to accomplish that, a means for separating plasma from whole blood “on demand” is needed, where sufficient plasma is drawn off for performing one reaction at a time so that as many assays as possible may be performed, in rank order of their clinical importance.
As has also been noted in the literature, elimination or reduction of diluent and reagent volumes results in an increase in sensitivity, but a satisfactory solution has not been presented for the problem of passively dispersing any inhomogeneities that occur during direct rehydration and mixing of dry reagents in a minimal sample liquid, where “passively” indicates an absence of mechanically assisted mixing.
In the case of kinetic assays, where a rate of a reaction under zero-order kinetic conditions is desired in order to measure the concentration of a selected analyte, what is needed is a device that can instantaneously mix a sample containing the rate-limiting analyte with all reactants and cofactors participating in the reaction and then, in a very short period of time during which the reaction rate is linear, before other reactants become rate limiting, measure a steady state reaction rate. This condition has not been met without use of a substantial sample or reagent volume taken up and manipulated in dedicated mixing circuitry powered by an external driver, for example as described by Lilja above, because without adequate mixing, the reaction rate is diffusion limited, not analyte limited, and therefore a rate or “kinetic” analysis cannot be achieved.
In view of the above, a need exists for a microfluidic device for conducting small-volume clinical assays by rapidly rehydrating and homogenizing dry reagents in a minimum of sample volume under passive mixing conditions, where the rate of reagent dissolution in sample is not so slow that analyte reaction kinetics cannot be measured. The present invention provides these and other features that will be apparent from the disclosure herein.